Chemical crosslinkers

ABSTRACT

Disclosed herein are methods of chemical conjugation comprising contacting a lysosomal enzyme with a first crosslinking agent to introduce aldehyde groups; contacting a lysosomal targeting peptide with a second crosslinking agent to introduce a hydrazide group at the N-terminal residue; contacting the lysosomal enzyme with aldehyde groups of step a. with the lysosomal targeting peptide with a hydrazide group at the N-terminal residue of step b; and forming a lysosomal enzyme-lysosomal targeting peptide conjugate.

This application claims the benefit of U.S. Ser. No. 61/794,784 filedMar. 15, 2013

FIELD OF THE INVENTION

The disclosed inventions relate generally to compositions and methodsfor chemical crosslinking.

BACKGROUND

Lysosomes are specialized intracellular organelles where proteins,various lipids (including glycolipids and cholesterol) and carbohydratesare degraded and recycled to their primary constituents that enablesynthesis of new proteins, membrane components and other molecules.Lysosomes are also utilized by cells to help maintain homeostasis andcellular health through an adaptive cellular process known as autophagythat increases lysosomal activity to provide additional amino acids forincreased biosynthesis of various proteins (e.g., antibodies andinterferons) and to supply nutrients for energy production to deal withstressful periods of nutrient deprivation or viral infections. Eachmetabolic process is catalyzed by a specific resident lysosomal enzyme.Genetic mutations can cause deficiencies in lysosomal biologicalactivities that alter metabolic processes and lead to clinical diseases.Lysosomal storage disorders (LSDs) are a class of approximately 50different human metabolic diseases caused by a deficiency for specificlysosomal proteins that results in the accumulation of varioussubstances within the endosomal/lysosomal compartments. Many of thesediseases have been well-characterized to understand the deficientlysosomal protein and the resultant metabolic defect. For example, thereare several LSDs of altered glycolipid catabolism such as Gaucher,Fabry, and Tay-Sachs/Sandhoff. Neimann-Pick C is characterized byimpaired lipid and cholesterol metabolism while diseases of alteredcarbohydrate metabolism such as glycogen storage diseases type II(Pompe) and type III (Corey-Forbes) have also been characterized. OtherLSDs alter metabolism of bone or extracellular matrices [e.g.,mucopolysaccharidoses (MPS I-VII), Gaucher] and protein turnover(neuronal ceroid lipofuscinoses; Batten, etc.). While LSDs arerelatively rare, they can cause severe chronic illness and often deathif not effectively treated.

There are no known cures for lysosomal storage diseases but a number ofdifferent treatment approaches have been investigated for various LSDsincluding bone marrow and umbilical cord blood transplantation, enzymereplacement therapy (ERT), substrate reduction therapy (SRT) andpharmacological chaperone therapy. Gene therapy is also being developedbut has not been tested clinically. Of these treatment approaches, ERTis the most established with multiple ERTs approved for the treatment ofvarious LSDs including Gaucher, Fabry, Pompe, MPS I, MPS II and MPS VIwhile one SRT drug is approved for the treatment of Gaucher disease.

The concept of ERT for the treatment of a lysosomal storage disease isfairly straightforward where a recombinant human lysosomal enzyme isadministered in patients to supplement the deficient biological activityand improve clinical symptoms. However, unlike other protein therapeutictreatments that function primarily at the cell surface or outside ofcells (e.g., anti-VEGF and other antibodies, erythropoietin, clottingfactors, etc.), lysosomal enzymes must function inside cells, withinlysosomes, and therefore use a mechanism for entering cells from theoutside and subsequent delivery to these internal compartments. Inmammals, the branched carbohydrate structures on the protein backbone oncertain asparagine residues (N-linked oligosaccharides; N-glycans) formost soluble lysosomal enzymes are post-translationally modified to forma specialized carbohydrate structure called mannose 6-phosphate (M6P).M6P is the natural biological signal for identification and transport ofnewly synthesized lysosomal proteins from the Golgi apparatus tolysosomes via membrane-bound M6P receptors. A class of M6P receptors(cation-independent M6P receptor; CI-MPR) also cycles to the plasmamembrane and is functionally active for binding and internalizingexogenous lysosomal proteins. The CI-MPR is believed to have evolved torecapture lysosomal proteins that escaped cells (via secretion out ofcells) and thus, provide a targeting mechanism for internalizingexogenous lysosomal proteins and is the basis for enzyme replacementtherapy for various LSDs.

Recombinant lysosomal enzyme replacement therapies have been shown to begenerally safe but their effectiveness for reducing clinical symptomsvaries widely. For example: Fabrazyme™ (recombinant acid α-galactosidaseA; Genzyme Corp.) ERT dosed at 1 mg/kg body weight every other week issufficient to clear accumulated substrate from endothelial cells inFabry disease while 40 mg/kg of Myozyme™ (recombinant human acidα-glucosidase, rhGAA; Genzyme Corp.) dosed every other week is onlymoderately effective for Pompe disease. The disparate efficacy isprimarily attributed to differences in the M6P content such that lowlevels of M6P correlates with poor drug targeting and lower efficacy.The manufacture of recombinant lysosomal enzymes is very challengingbecause it is extremely difficult to control carbohydrate processing,particularly the level of M6P in mammalian expression systems. Twospecialized Golgi enzymes catalyze the M6P modification;N-acetylglucosamine phosphotransferase adds phosphate-linkedN-acetylglucosamine onto certain terminal mannose residues whileN-Acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (alsoknown as Uncovering Enzyme) removes the covering N-acetylglucosamine toreveal the M6P signal. However, N-acetylglucosamine phosphotransferaseis limiting in cells and this biochemical reaction is inherentlyinefficient for various lysosomal proteins. Over-expression of lysosomalproteins during the manufacturing process greatly exacerbates thisproblem and leads to highly variable amounts of M6P. Consequently,carbohydrate processing is typically incomplete and leads to theproduction of recombinant lysosomal enzymes with mixtures of N-glycansthat contain M6P, non-M6P structures of high-mannose type N-glycans andcomplex-type N-glycans (typical for secretory proteins). To complicatematters, dead or damaged cells release enzymes such as phosphatases intothe cell culture medium which remove M6P. Consequently, reduced M6Pcontent lowers the binding affinity of a recombinant lysosomal enzymefor M6P receptors and decreases its cellular uptake and thereby, reducedrug efficacy. Dead or damaged cells release other glycosidases thatremove other carbohydrates (e.g., sialic acids, galactose, etc.) toreveal internal carbohydrates that are not typically exposed and theseN-glycans are readily identified as aberrant. These incomplete N-glycanstructures increase the clearance rate of recombinant lysosomal proteinsfrom the circulation which can also reduce drug efficacy. Higher drugdoses are therefore necessary to compensate for reduced efficacy. Higherdrug dose requirements however have multiple negative implications: (1)higher drug dose could be cost-prohibitive by increasing an alreadyexpensive treatment; (2) high drug doses require long infusion times;(3) large amounts of circulating drug results in significant antibodyresponses (seen in most Pompe patients) and numerous patients have alsoexperienced allergic reactions during infusions. The FDA has issued a“black-label warning” for Myozyme and the drug is typically administeredvery slowly at the beginning but ramped up over the course of theinfusion. This strategy helps to mitigate the allergic responses butsignificantly lengthens infusion times where 12-hr infusions are notuncommon.

One potential strategy for improving drug targeting for variouslysosomal ERTs employs a targeting peptide to efficiently target ERTs tolysosomes without requiring the traditional M6P carbohydrate structures.This is conceptually feasible since the cation-independent M6P receptorcontains a distinct binding domain for a small peptide calledinsulin-like growth factor 2 (IGF-2) and this receptor is thereforeknown as the IGF-2/(IGF-2/CI-MPR). This receptor is in fact solelyresponsible for internalizing exogenous M6P-bearing lysosomal proteinsbecause the IGF-2/CI-MPR is present and biologically active on the cellsurface. The other class of M6P receptors, the cation-dependent M6Preceptor (CD-MPR), is only involved in the transport of lysosomalproteins within cells because it is not biologically active on cellsurfaces and lacks the IGF-2 peptide binding domain. The IGF-2/CI-MPRhas two separate binding sites for M6P (domains 1-3 and 7-9,respectively) such that it binds a mono-M6P N-glycan (1 M6P residue onN-glycan) with moderate affinity or a bis-M6P N-glycan (two M6P residueson the same N-glycan) with approximately 3000-fold higher affinity.Since lysosomal proteins contain mixtures of complex (no M6P), mono- andbis-M6P N-glycans, their affinities for the IGF-2/CI-MPR vary widelydepending on the type and amount of M6P-bearing N-glycans.

There remains a need to develop strategies to create IGF-2-linkedproteins for improved protein targeting. There remains a need forconstructs that maintain the correct protein conformation.

SUMMARY OF THE INVENTION

Disclosed herein are methods of chemical conjugation comprising a)contacting a lysosomal enzyme with a first crosslinking agent tointroduce aldehyde groups; b) contacting a lysosomal targeting peptidewith a second crosslinking agent to introduce a hydrazide group at theN-terminal residue; contacting the lysosomal enzyme with aldehyde groupsof step a) with the lysosomal targeting peptide with a hydrazide groupat the N-terminal residue of step b); and forming a lysosomalenzyme-lysosomal targeting peptide conjugate.

Also provided herein are bifunctional crosslinkers comprising acetone-,dimethylbenzyloxycarbonyl-, trimethylbenzyloxycarbonyl-protectedhydrazide groups, or any combination thereof. Also provided herein aremodified lysosomal targeting peptides comprising a protected hydrazidegroup.

Disclosed herein are methods of making a modified lysosomal targetingpeptide, comprising: contacting a lysosomal targeting peptide with acrosslinking agent to introduce a hydrazide group at the N-terminalresidue. Also provided are methods of linking one or more lysosomaltargeting peptides to a lysosomal enzyme comprising deprotecting ahydrazide-modified lysosomal targeting peptide in solution to form adeprotected hydrazide-modified lysosomal targeting peptide; and linkingat least one deprotected hydrazide modified lysosomal targeting peptideto a lysosomal enzyme.

Disclosed herein are lysosomal enzyme-lysosomal targeting peptideconjugates, comprising: a lysosomal enzyme crosslinked to one or morelysosomal targeting peptides via one or more bifunctional crosslinkers.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further understood when read in conjunctionwith the appended drawings. For the purpose of illustrating the subjectmatter, there are shown in the drawings exemplary embodiments of thesubject matter; however, the presently disclosed subject matter is notlimited to the specific methods, devices, and systems disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 shows the modification of the vIGF2 peptide as monitored on anHPLC system.

FIG. 2 shows the vIGF2-GAA conjugate's ability to bind to theIGF2-CI-MPR receptor in binding plate assays.

FIG. 3 shows the chemical structures of some of the disclosedcrosslinkers.

FIG. 4. (A) shows the retention time for vIGF2 peptide upon attachmentof tBoc-protected hydrazide linker (middle panel) relative to thestarting unmodified vIGF2 peptide (upper panel) and removal of the tBocprotection group was shown to cause another shift (decrease) in theretention on C4 RP-HPLC (lower panel). (B) shows the results of vIGF2peptide-conjugated rhGAA (via resultant hydrazone linkage) in anIGF2/CI-MPR receptor plate binding assays.

FIG. 5 shows the evaluation of cellular uptake for hydrazone linkedvIGF2-rhGAA by internalization of exogenous unconjugated rhGAA (10-500nM) and vIGF2-rhGAA (2-50 nM) in L6 rat skeletal muscle myoblasts. (A).vIGF2-rhGAA was shown to be internalized substantially better thanunconjugated rhGAA in L6 myoblasts at all protein concentrations tested.(B) L6 myoblast lysates were analyzed by Western blotting using rabbitanti-human GAA polyclonal primary antibodies.

FIG. 6 shows retention time in a C4 RP-HPLC assay of purified vIGF2peptide chemically modified with a 20-fold molar excess of thebifunctional crosslinker methylbenzyloxy carbonyl (BOM)-protectedhydrazide (NHS-PEG4-BOM-hydrazide) to introduce BOM-protected hydrazidefunctional group at N-terminus of peptide.

FIG. 7. (A) shows retention time in a C4 RP-HPLC assay of purified vIGF2peptide chemically modified with a 20-fold molar excess of thebifunctional crosslinker Phthalimidooxy-PEG12-NHS ester to introduce aphthalimidooxy functional group at N-terminus of peptide. (B) showsaffinity assays of oxime-linked vIGF2-rhGAA in an IGF2/CI-MPR receptorplate binding assays to determine whether attached vIGF2 peptideimproved rhGAA affinity for the IGF2/CI-MPR receptor.

FIG. 8 shows retention time in a C4 RP-HPLC assay of purified vIGF2peptide chemically modified with a 20-fold molar excess of thebifunctional crosslinker tBoc-aminooxy-PEG₁₂-PFB to introduce atBoc-protected aminooxy functional group at N-terminus of peptide.

FIG. 9 shows IGF2/CI-MPR receptor plate binding assays ofaminooxy-modified vIGF2 peptide directly conjugated to chemicallyoxidized rhGAA (containing chemically reactive aldehyde groups oncarbohydrates) to form the resultant oxime linkages.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present subject matter can be understood more readily by referenceto the following detailed description which forms a part of thisdisclosure. It is to be understood that this invention is not limited tothe specific products, methods, conditions or parameters describedand/or shown herein, and that the terminology used herein is for thepurpose of describing particular embodiments by way of example only andis not intended to be limiting of the claimed invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

As employed above and throughout the disclosure, the following terms andabbreviations, unless otherwise indicated, shall be understood to havethe following meanings.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “acompound” is a reference to one or more of such compounds andequivalents thereof known to those skilled in the art, and so forth. Theterm “plurality”, as used herein, means more than one. When a range ofvalues is expressed, another embodiment includes from the one particularand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it isunderstood that the particular value forms another embodiment. Allranges are inclusive and combinable.

The chemical conjugation approach can be improved by novel bifunctionalcrosslinkers which can be utilized for modification of vIGF2 peptide tointroduce a protected hydrazide group. The acid labile protecting groupscan be efficiently removed using mild acidic buffers for better recoveryof modified vIGF2 peptide. The deprotected hydrazide-modified vIGF2peptide can then be lyophilized and stored as a dried powder to preservethe chemical reactivity of the hydrazide moiety. The deprotectedhydrazide-modified vIGF2 peptide can be stored indefinitely untilchemical coupling to lysosomal enzymes. These novel crosslinkers cantherefore provide significantly more stable components and bettercontrol of chemical conjugation process to enable scale up of process.

This chemical conjugation approach involves modifying the amino(N)-terminus and one or more lysine residues on a recombinant humanlysosomal enzyme using a first crosslinking agent to introduce novelacetone-protected hydrazide groups on recombinant human lysosomalenzymes. The first crosslinking agent modified recombinant humanlysosomal enzyme can then be purified to remove excess crosslinkers andreaction byproducts. The purification can be performed using acidic pHbuffers which preserves catalytic activity for lysosomal enzyme anddisplaces the acetone protecting group to expose the chemically reactivehydrazide groups. Separately, the amino (N)-terminus of a shortextension linker region preceding vIGF2 peptide can be modified using asecond crosslinking agent to introduce novel chemically reactivealdehyde group on vIGF2 peptide. The modified vIGF2 peptide can then bepurified to remove excess crosslinkers and reaction byproducts and thenlyophilized. In a final coupling reaction, the hydrazide-modifiedrecombinant human lysosomal enzyme can be added directly to thelyophilized aldehyde-modified vIGF2 peptide to generate the vIGF2-enzymeconjugate. While this method is effective to generate vIGF2-enzymeconjugates, the final chemical conjugation reaction is preferablyperformed rapidly because the introduced hydrazide groups are not stablein aqueous solutions and thus, the chemical reactivity is diminishedover a relatively short time (within 1-2 days). The scale up of processcan therefore be challenging if the modification of lysosomal enzymeswith bifunctional crosslinker and purification of hydrazide-modifiedlysosomal enzymes cannot be quickly completed (preferably within 1 day).

It would be preferable to change the order of modification whereby thelysosomal enzyme can be modified with a crosslinking agent to introducealdehyde groups and the vIGF2 peptide would be modified with acrosslinking agent to introduce hydrazide. However, a majority ofhydrazide-containing crosslinkers such as succinimidyl6-hydrazinonicotinate acetone (S-Hynic) or related crosslinkers causeaggregation and/or precipitation of vIGF2 peptide and lead tosignificant loss of peptide. Appropriate crosslinkers and methods tomodify vIGF2 peptide with bifunctional crosslinkers to introduce aprotected hydrazide group on vIGF2 peptide are provided herein. Theprotecting group can be subsequently removed by incubation in acidicbuffers and the hydrazide-modified vIGF2 peptide can be purified byreverse phase chromatography on HPLC. The purified hydrazide-modifiedvIGF2 peptide can then be lyophilized and stored as a dried powder. Thisstrategy ensures that the hydrazide group remains chemically active forcoupling to lysosomal enzymes. In a separate reaction, a recombinantlysosomal enzyme can be modified with a crosslinking agent to introducenovel aldehyde groups. After purification to remove excess crosslinkerand reaction byproducts, the aldehyde-modified lysosomal enzymes canthen be stored until coupling to vIGF2 peptide because the introducedaldehyde groups are chemically stable in aqueous solutions. This newstrategy provides “hold steps” in the process where individualcomponents (modified lysosomal enzyme and modified vIGF2) can be storedindefinitely until coupling to generate the vIGF2-enzyme conjugates.This new method therefore generates the individual components withsubstantially higher stability and provides better overall control ofconjugation process which is conducive to scale up of process.

Suitable methods of chemical conjugation can comprise a) contacting alysosomal enzyme with a first crosslinking agent to introduce aldehydegroups; b) contacting a lysosomal targeting peptide with a secondcrosslinking agent to introduce a hydrazide group at the N-terminalresidue; contacting the lysosomal enzyme with aldehyde groups of step a)with the lysosomal targeting peptide with a hydrazide group at theN-terminal residue of step b); and forming a lysosomal enzyme-lysosomaltargeting peptide conjugate.

The lysosomal targeting peptide may suitably comprise variantinsulin-like growth factor 2 (vIGF2), preferably a variant of humaninsulin-like growth factor 2. In one embodiment, such a variant IGF 2may include amino acid deletions and substitutions that permit vIGF2peptide to maintain high affinity for the IGF-2/CI-MPR while reducingpeptide binding affinity to IGF-1 and Insulin receptors. For example,the deletions and substitutions may comprises one or more of thefollowing changes: deletion of N-terminal amino acid residues 1-4 sincethese residues are not needed for binding intended IGF2/CI-MPR receptorand also eliminates a proline at position 4 and the associated bend orkink in the protein at the N-terminus; glutamic acid residue at position6 of wildtype human IGF2 substituted with arginine for reducing oreliminating peptide binding to serum IGF binding proteins (IGFBPs),tyrosine residue of wildtype human IGF 2 at position 27 substituted withleucine for reducing or eliminating peptide binding the insulin andIGF-1 receptors, and the lysine residue at position 65 of human IGF-2substituted with arginine to prevent chemical modification of peptide atthat position.

In still further embodiments, the variant IGF2 can include or be one ofthe following sequences:

SEQ ID NO: 1 AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE SEQ ID NO: 2SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDL ALLETYCATPARSESEQ ID NO: 3 GGGGSRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE SEQ ID NO: 4GGGGSGGGGSRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE

Suitable first crosslinking agents includeN-succinimidyl-4-formylbenzamide (S-4FB). A suitable second crosslinkingagent includes N-tert-butoxycarbonyl (tBoc)-protected hydrazidecrosslinker (NHS-PEG4-tBoc-hydrazide) or a methoxybenzyloxy carbonyl(BOM)-protected hydrazide (NHS-PEG4-BOM-hydrazide). In other suitableembodiments the second crosslinking agent can include acetone-,dimethylbenzyloxycarbonyl-, trimethylbenzyloxycarbonyl-protectedhydrazide groups, or any combination thereof. In other suitableembodiments the bifunctional crosslinker can include acetone-protectedNHS-PEG4-hydrazide, dimethylbenzylcarbonyl-protected NHS-PEG4-hydrazide,trimethylbenzyloxycarbonyl-protected NHS-PEG4-hydrazide, or anycombination thereof

Suitable lysosomal enzymes include acid α-glucosidase (rhGAA), acidα-galactosidase A (GLA), acid β-glucuronidase (GUS), acid α-iduronidaseA (IduA), acid iduronidate 2-sulfatase (I2S), β-hexosaminidase A (HexA),β-hexosaminidase B (HexB), acid α-mannosidase A, β-glucocerebrosidase(GlcCerase), acid lipase (LPA), or any combination thereof. In othersuitable embodiments the lysosomal enzyme can be mammalian and themammalian lysosomal enzyme can be human.

The lysosomal targeting peptide with a hydrazide group at the N-terminalresidue is typically added to the lysosomal enzyme at about four-foldmolar excess. Aniline can also be suitably added at that time. Themethods provided herein can further comprise one or more purificationsteps of the lysosomal enzyme-lysosomal targeting peptide conjugate. Forexample, purification can be carried out using size-exclusionchromatography.

The methods provided herein can further comprise evaluating thelysosomal enzyme-lysosomal targeting peptide conjugate's ability to bindthe IGF2/CI-MPR receptor. Binding of the lysosomal enzyme-lysosomaltargeting peptide conjugate typically saturates at or above about 25 nMof lysosomal enzyme-lysosomal targeting peptide conjugate. Accordingly,the lysosomal enzyme-lysosomal targeting peptide conjugate can maintainthe correct protein confirmation by enabling high affinity binding tothe IGF2/CI-MPR receptor.

Suitable bifunctional crosslinkers can comprise acetone-,dimethylbenzyloxycarbonyl-, trimethylbenzyloxycarbonyl-protectedhydrazide groups, or any combination thereof. In other suitableembodiments the bifunctional crosslinker can comprise Acetone-protectedNHS-PEG4-hydrazide, Dimethylbenzylcarbonyl-protected NHS-PEG4-hydrazide,Trimethylbenzyloxycarbonyl-protected NHS-PEG4-hydrazide, or anycombination thereof.

Suitable modified lysosomal targeting peptides can comprise a protectedhydrazide group. In other suitable embodiments the modified lysosomaltargeting peptide can comprise modified vIGF2 peptide. In other suitableembodiments the hydrazide group is protected by acetone-,dimethylbenzyloxycarbonyl-, trimethylbenzyloxycarbonyl-groups, or anycombination thereof. In other suitable embodiments the modifiedlysosomal targeting peptide is in a powder. In other suitableembodiments the modified lysosomal targeting peptide is lyophilized.

Suitable methods of making a modified lysosomal targeting peptideinclude contacting a lysosomal targeting peptide with a crosslinkingagent to introduce a hydrazide group at the N-terminal residue. Asuitable lysosomal targeting peptide is vIGF2. A suitable crosslinkingagent includes N-tert-butoxycarbonyl (tBoc)-protected hydrazidecrosslinker (NHS-PEG4-tBoc-hydrazide). The crosslinking agent cancomprise acetone-, dimethylbenzyloxycarbonyl-,trimethylbenzyloxycarbonyl-protected hydrazide groups, or anycombination thereof. The bifunctional crosslinker can comprisesacetone-protected NHS-PEG4-hydrazide, dimethylbenzylcarbonyl-protectedNHS-PEG4-hydrazide, trimethylbenzyloxycarbonyl-protectedNHS-PEG4-hydrazide, or any combination thereof. The methods providedherein can further comprise deprotecting the protected hydrazide groupsof the modified lysosomal targeting peptide. As well, a further step oflyophilizing the modified lysosomal targeting peptide can be implementedto form a powder.

Suitable methods of linking one or more lysosomal targeting peptides toa lysosomal enzyme can comprise deprotecting a hydrazide-modifiedlysosomal targeting peptide in solution to form a deprotectedhydrazide-modified lysosomal targeting peptide; and linking at least onedeprotected hydrazine modified lysosomal targeting peptide to alysosomal enzyme. The lysosomal targeting peptide can comprise vIGF2,and the lysosomal enzyme can comprise acid α-glucosidase (rhGAA), acidα-galactosidase A (GLA), acid β-glucuronidase (GUS), acid α-iduronidaseA (IduA), acid iduronidate 2-sulfatase (I2S), β-hexosaminidase A (HexA),β-hexosaminidase B (HexB), acid α-mannosidase A, β-glucocerebrosidase(GlcCerase), acid lipase (LPA), or any combination thereof

Suitable lysosomal enzyme-lysosomal targeting peptide conjugates cancomprise a lysosomal enzyme crosslinked to one or more lysosomaltargeting peptides (e.g., vIGF2) via one or more bifunctionalcrosslinkers. Suitable bifunctional crosslinkers can compriseacetone-protected NHS-PEG4-hydrazide, dimethylbenzylcarbonyl-protectedNHS-PEG4-hydrazide, trimethylbenzyloxycarbonyl-protectedNHS-PEG4-hydrazide, N-succinimidyl-4-formylbenzamide (S-4FB), or anycombination thereof. The lysosomal enzyme suitably comprises acidα-glucosidase (rhGAA), acid α-galactosidase A (GLA), acidβ-glucuronidase (GUS), acid α-iduronidase A (IduA), acid iduronidate2-sulfatase (I2S), β-hexosaminidase A (HexA), β-hexosaminidase B (HexB),acid α-mannosidase A, β-glucocerebrosidase (GlcCerase), acid lipase(LPA), or any combination thereof

EXAMPLES

The following examples, while illustrative individual embodiments, arenot intended to limit the scope of the described invention, and thereader should not interpret them in this way.

Example 1

A new chemical conjugation approach was utilized for chemical couplingof vIGF2 peptide onto lysosomal enzymes to increase their binding to theIGF2/CI-MPR receptor. This approach is different from what had beenpreviously described such that the lysosomal enzymes are modified with afirst crosslinking agent to introduce aldehyde groups while vIGF2peptide is modified with a second crosslinking agent to introduce ahydrazide group at the N-terminal residue. Specifically, recombinantwildtype human acid α-glucosidase (rhGAA) was concentrated to 7.5 mg/mlvia a concentrator device equipped with a 50 kDa molecular weight cutoffmembrane and then buffer exchanged into 50 mM sodium phosphate (pH6.5)/100 mM NaCl/0.05% polysorbate-80 by dialysis at 4° C. overnight.rhGAA was then chemically modified with a 20-fold molar excess ofN-succinimidyl-4-formylbenzamide (S-4FB; Solulink) crosslinker for 2 hrsat ambient temperature to introduce novel benzaldehyde groups. Thebenzaldehyde-modified rhGAA is then purified in 50 mM NaOAc (pH 4.8)/50mM NaCl buffer to remove excess crosslinker and reaction byproducts andstored at 4° C. until chemical coupling to vIGF2 peptide.

In a separate reaction, purified vIGF2 peptide was reconstituted at 0.6mg/ml in 50 mM sodium phosphate (pH 7.5)/50 mM NaCl/0.05% polysorbate-80and modified with a 20- to 40-fold molar excess of N-tert-butoxycarbonyl(tBoc)-protected hydrazide crosslinker (NHS-PEG4-tBoc-hydrazide; QuantaBiodesign). The modification of vIGF2 peptide was monitored using ausing a 4.6×250 mm, 5 μm, 300 Å C4 Jupiter column (Phenomenex, Torrence,Calif.) on an Agilent Technologies (Palo Alto, Calif.) HPLC system (C4RP-HPLC) as shown in FIG. 1. Mobile phases consisted of Buffer A (0.1%TFA in water) and Buffer B [0.1% TFA in acetonitrile (MeCN)]. Thisanalytical method, designated as C4 reverse phase HPLC (C4 RP-HPLC), wasutilized to characterize IGF2 peptide samples by injecting 5-100 μLsamples on the C4 column pre-equilibrated with 74% Buffer A, 26% BufferB at 2 mL/min Two minutes after sample injection, a linear gradient of26% to 33% Buffer B was developed over 13 minutes. Under theseconditions, unmodified vIGF2 peptide elutes at approximately 11.5minutes. As the crosslinker is chemically attached to vIGF2 during themodification reaction, the modified IGF2 peptide has a distinctchromatographic profile and appears as a new later eluting peak on C4RP-HPLC with an apparent retention time of ˜13.5 min. When vIGF2 peptidewas >95% modified with crosslinker, the reaction was halted by addingTFA (15% final concentration) and incubated for up to 4 hrs. Thisincubation step was used to remove the tBoc protecting group asevidenced by the significant shift in the retention time of vIGF2 on C4RP-HPLC. Under these experimental conditions, approximately 50% of thetBoc was removed. Significant loss of the modified vIGF2 peptideoccurred when using longer incubations or higher TFA concentrations. Thehydrazide-modified vIGF2 peptide was then purified via a preparative C4RP-HPLC, lyophilized and stored as a dried powder and stored at 4° C.until chemical coupling to rhGAA.

The benzaldehyde-modified rhGAA was chemically conjugated to thedeprotected hydrazide-modified vIGF2 peptide in a final reaction byadding the lysosomal enzyme directly to the lyophilized peptide at a4-fold molar excess of peptide. Aniline, a chemical catalyst of thisreaction, was added to a final concentration of 10 mM and the reactionwas incubated overnight at ambient temperature with gentle rocking. ThevIGF2-rhGAA conjugate was purified the following day using sizeexclusion chromatography to remove excess vIGF2 peptide using 50 mMsodium phosphate (pH 6.2)/100 mM NaCl/0.05% (v/v) polysorbate-80 buffer.

To determine whether the modified vIGF2 peptide retained the correctprotein structure after removal of the tBoc protecting group, thevIGF2-GAA conjugate was evaluated for its ability to bind theIGF2/CI-MPR receptor in plate binding assays as shown in FIG. 2.Briefly, unconjugated rhGAA and vIGF2-rhGAA was serially diluted withBinding Buffer [40 mM HEPES (pH 6.7), 150 mM NaCl, 10 mM EDTA and 0.02%(v/v) Tween-20] to obtain final GAA concentrations ranging from 0.01-10μg/ml and incubated in IGF2/CI-MPR receptor coated plates at 3TC for 30min. The receptor plate was washed 3× with Binding Buffer to removeunbound enzyme and the amount of bound GAA enzyme was measured using thefluorogenic 4-methylumbeliferyl-α-glucose substrate[4-MU-α-Glc; 1 mMfinal substrate concentration in 50 μl 0.1M NaOAc (pH 4.8)]. Forty fiveμL of the reaction samples were then transferred to new 96-well black,clear bottom assay plates and 125 μL 0.5 M NaOH was added to stop theenzymatic reaction and to raise the pH. The liberated 4-MU fluorescencefrom the individual enzymatic reactions was then quantified in afluorescence plate reader (using 370 nm excitation and 460 emissionwavelengths, respectively). As shown in FIG. 2, vIGF2-GAA bound theIGF2/CI-MPR receptor substantially better than the rhGAA startingmaterial at all concentrations tested. The binding of vIGF2-GAA appearedto be saturated at or above 25 nM as expected. These results thereforeshow that vIGF2 peptide was able to maintain the correct proteinconformation after removal of the tBoc deprotection group which enabledhigh affinity binding to the IGF2/CI-MPR receptor.

Example 2

The NHS-PEG4-tBoc-hydrazide crosslinker was shown to efficiently modifyvIGF2 peptide to introduce novel hydrazide group while maintaining goodsolubility for the peptide. However, the tBoc protecting group wasdifficult to remove using mild acid conditions and used highconcentrations of TFA (15%) and 4-hr incubations for deprotection. Underthese harsh conditions, we were still only able to recover ˜50% of thedeprotected hydrazide-modified vIGF2 peptide. The approach would befurther improved if crosslinkers can be designed with more labileprotecting groups for efficient removal and better recovery of modifiedpeptide.

Novel bifunctional crosslinkers have been developed for efficientattachment to vIGF2 peptide and to introduce protected hydrazide groupswhile maintaining good solubility for peptide. These new crosslinkerscontain acetone-, dimethylbenzyloxycarbonyl-, ortrimethylbenzyloxycarbonyl-protected hydrazide groups as shown in FIG.3. These protecting groups are much more labile than tBoc and would usemild acid conditions (e.g., <5% TFA) for efficient removal and highrecovery of the modified peptide. Moreover, these new crosslinkersshould permit long-term storage of the deprotected modified vIGF2peptide after purification and lyophilization. These new crosslinkerstherefore will enable scale up and better control of the chemicalconjugation process.

Example 3

Recombinant human acid α-glucosidase (rhGAA) was concentrated to 8-10mg/ml at small scale (i.e., <100 mg) using centrifugal concentratordevices (e.g., Amicon Ultra with 50 kDa nominal molecular weight cutoff(MWCO) membrane; Millipore) and then buffer exchanged into ModificationBuffer [50 mM sodium phosphate (pH 6.5)/100 mM NaCl/100 mM glucose/2%mannitol/0.05% polysorbate-80] via dialysis overnight at 4° C. Forlarger scale preparations (e.g., >200 mg rhGAA), protein concentrationand buffer exchange can be achieved by diafiltration/concentration usinga tangential flow filtration (TFF) system with a 50 kDa MWCO membrane.rhGAA protein concentration is determined by UV absorbance spectroscopyat 280 nm using the molar extinction coefficient (ε) of 166,117 M⁻¹cm⁻¹. The rhGAA protein concentration was then adjusted to 7.5 mg/ml bydilution with Modification Buffer and incubated with a 25-fold molarexcess of the bifunctional crosslinker succinimidyl 4-formylbenzoate(SFB) at about 20° C. for 4 hours to introduce novel benzaldehyde(aromatic aldehyde) groups on rhGAA. Benzaldehyde-modified rhGAA wasdialyzed against 50 mM NaOAc (pH 4.8)/100 mM NaCl/0.05% polysorbate-80at 4° C. with multiple changes of buffer to remove excess crosslinkerand reaction byproducts. The chemically modified rhGAA is stable in thisbuffer and can be stored indefinitely until chemical conjugation tovIGF2 peptide. Importantly, the introduced benzaldehyde groups on rhGAAremain chemically reactive for conjugation to hydrazide- oraminooxy-modified vIGF2 peptide. We have also determined thatbenzaldehyde-modified rhGAA can be stored in 50 mM sodium phosphate (pH6.0)/100 mM NaCl/0.05% polysorbate-80 with no adverse effects on GAAstability or coupling efficiency.

To determine the extent of chemical modification and to identify thespecific amino acid residues that are modified, peptide maps from thebenzaldehyde-modified rhGAA were generated and directly compared to thestarting unmodified starting rhGAA sample by liquid chromatography/massspectroscopy analysis (LC-MS). Briefly, a reference peptide map for thestarting unmodified rhGAA sample was generated by digesting rhGAA intopeptide fragments using a mixture of specific proteases followed by C18reverse phase high performance liquid chromatography and analysis ofindividual peptide fragments by mass spectroscopy. Four differentpreparations of benzaldehyde-modified rhGAA were digested and processedin the same manner and compared to the reference peptide map forunmodified starting rhGAA. Our LC-MS results indicate that an average ofapproximately 2 novel benzaldehyde groups is introduced on rhGAA usingthis chemical modification procedure. Moreover, the LC-MS results showthat chemical modification of rhGAA is not randomly distributed acrossthe different lysine residues in rhGAA. Rather, chemical modification ofrhGAA appears to be much more ordered and confined to a few lysineresidues as shown in Table 1. The LC-MS data show that ˜90% of Lys⁹⁰² ismodified with the benzaldehyde moiety while approximately 50% of Lys⁷³²and Lys⁸³⁸ are modified. A significantly smaller fraction of Lys¹¹³ (8%)and Lys¹⁶¹ (17%) are modified under these experimental conditions. Theexperimental error for this LC-MS analysis was estimated to be ±10%.

TABLE 1 Identification of chemically modified amino acid residues inrhGAA Residues Modified Estimated Modified GAA Batch Lys¹¹³ Lys¹⁶¹Lys⁷³² Lys⁸⁴⁸ Lys⁹⁰² Residues A 8% 17% 53% 40% 90% 2.11 (Feb. 1, 2013) B9% 16% 52% 45% 89% 2.14 (Apr. 4, 2013) C 8% 17% 53% 40% 90% 2.11 (Apr.9, 2013) D 6% 17% 53% 43% 89% 2.13 (Apr. 12, 2013) AVE 8 ± 1.3% 17 ±0.6% 53% ± 0.6% 42% ± 2.4% 89% ± 0.5% 2.12

The LC-MS data were informative and have very important implications forthe described chemical conjugation approach. There are 15 differentlysine residues within rhGAA; each theoretically has the same potentialfor chemical modification. Our empirical data however are contrary tothis hypothesis and show that the chemical modification process appearsto be much more ordered than anticipated wherein only a few, selectlysine residues were consistently modified with benzaldehyde groups for4 different batches of SFB-modified rhGAA using the method describedherein. Moreover, the LC-MS data indicate that there is preferentialmodification of lysine residues such that Lys⁹⁰² is always modified withcrosslinker while approximately half of Lys⁷³² and Lys⁸³⁸ residues aremodified with crosslinker. Only a small fraction of Lys¹¹³ and Lys¹⁶¹were modified with benzaldehyde group. These data suggest that theobserved preferential modification was dependent on the accessibilityand chemical competency of individual lysine residues for chemicalmodification with crosslinker. For example, lysine residues may beprotonated and involved with forming salt bridges (i.e., electrostaticinteractions) with negatively charged aspartic acid or glutamic acidresidues and not available for chemical modification. The LC-MS datasuggest that Lys⁹⁰² is the most accessible and chemically competent formodification and likely the likely the first residue modified. Further,Lys⁷³² and Lys⁸³⁸ are less accessible than Lys⁹⁰² for chemicalmodification but Lys⁷³² and Lys⁸³⁸ have similar accessibility relativeto each other since an approximately equivalent fraction of each ismodified. These data suggest that either Lys⁷³² or Lys⁸³⁸ is the secondlysine residue modified. Lys¹¹³ and Lys¹⁶¹ are only partially accessiblefor chemical modification such that only a small fraction of rhGAAcontains benzaldehyde at these lysine residues. No chemical modificationof the amino (N-) terminus was observed for rhGAA but this enzyme isknown to be naturally modified (cyclized) in cells to form pyroglutamatethat is not chemically competent for modification with bifunctionalcrosslinkers.

These collective data indicate that the described chemical modificationprocedure can be utilized to reproducibly yield rhGAA with an average of2.12 introduced benzaldehyde groups for coupling to vIGF2 peptide. Sincethe number of vIGF2 peptide conjugated to rhGAA is dependent on thenumber of introduced benzaldehyde groups, these results indicate that anaverage of 2 vIGF2 peptides can be conjugated to rhGAA.

After chemical modification with SFB, benzaldehyde-modified rhGAA isvery stable in slightly acidic buffers such as 50 mM sodium phosphate(pH 6.0)/100 mM NaCl/0.05% polysorbate-80 or 50 mM NaOAc (pH 4.8)/100 mMNaCl/0.05% polysorbate-80 and can be stored long-term prior to chemicalconjugation which is highly desirable to enable scale up of thisconjugation process.

Example 4

Benzaldehyde-modified rhGAA can be utilized for chemical conjugation tovIGF2 peptide via specific chemical groups such as hydrazide to form ahydrazone linkage as follows. Lyophilized, purified vIGF2 peptide wasreconstituted at 0.6 mg/ml in 50 mM sodium phosphate (pH 7.5)/50 mMNaCl/0.05% polysorbate-80 and incubated with a 20-fold molar excess ofthe bifunctional crosslinker N-tert-butoxycarbonyl (tBoc)-protectedhydrazide crosslinker (NHS-PEG4-tBoc-hydrazide; Quanta Biodesign) atambient temperature to introduce a novel tBoc-protected hydrazide groupat the N-terminus. The reaction was monitored by C4 reverse phase highperformance liquid chromatography (C4 RP-HPLC) to assess the progressionand extent of modification of vIGF2. C4 RP-HPLC method utilized a4.6×250 mm 5 μm, 300 Å C4 Jupiter column (Phenomenex, Torrence, Calif.)on an Agilent Technologies (Palo Alto, Calif.) HPLC system consisting ofa 1100 series quaternary pump, automated sampler, thermo stated columncompartment and diode-array detector (DAD) with Agilent ChemStationSoftware (Rev. B.04.03). Mobile phases consisted of Buffer A: 0.1%trifluoracetic acid (TFA) in water and Buffer B: 0.1% TFA inacetonitrile (MeCN). Peptide samples were loaded onto C4 columnpre-equilibrated with 26% acetonitrile (MeCN/0.1% TFA). After 2 minutes,the column was developed using a 26-33% linear gradient of MeCN/0.1% TFAover 13 minutes. As shown in FIG. 4A, a significant shift (increase) inthe retention time is observed upon chemical modification of vIGF2peptide to attach PEG4-tBoc-hydrazide (middle panel). After completionof chemical modification of vIGF2 peptide (typically by 2 hrs),tBoc-hydrazide-modified vIGF2 peptide was purified by preparative C4RP-HPLC to remove excess crosslinker and reaction byproducts andlyophilized to remove volatile solvents. The dried peptide was thenreconstituted in 2% TFA (in dH₂O) and incubated at ambient temperaturefor up to 72 hrs to remove the tBoc protection group and exposehydrazide group for subsequent conjugation to lysosomal enzymes. Theprogression of tBoc de-protection was also monitored by C4 RP-HPLC andtypically shown to decrease the retention time of peptide upon removalof tBoc to near that of the starting, unmodified vIGF2 peptide (FIG. 4A,bottom panel). Complete de-protection of tBoc group typically requiredapproximately 60 hrs under these experimental conditions. tBocde-protected hydrazide-modified vIGF2 was then purified by preparativeC4 RP-HPLC, lyophilized and stored dry until chemical conjugation tobenzaldehyde-modified rhGAA. Removal of the tBoc protecting group hasalso been tested using higher TFA concentrations (e.g., 4% TFA) andother acids and organic solvents but those conditions led to much lowerrecovery of hydrazide-modified vIGF2 peptide. De-protected hydrazidevIGF2 peptide was chemically conjugated to benzaldehyde-modified rhGAA(using 4-fold molar excess vIGF2 peptide to rhGAA) via a resultanthydrazone linkage as follows. The protein concentration ofbenzaldehyde-modified rhGAA was typically adjusted to 4-5 mg/ml with 50mM sodium phosphate (pH 6.0)/100 mM NaCl/0.05% polysorbate-80 buffer andthe enzyme was added directly to lyophilized de-protectedhydrazide-modified vIGF2 peptide (at a molar ratio of 1 mole rhGAA:4moles vIGF2 peptide) and incubated at about 20° C. for approximately 16hours. Aniline (5-10 mM) can also be added to increase the rate ofcoupling but we have found that it was not absolutely required forcoupling under these experimental conditions. vIGF2 peptide-conjugatedrhGAA was then purified by size exclusion chromatography in 50 mM sodiumphosphate (pH 6.0)/100 mM NaCl/2% mannitol/0.05% polysorbate-80 bufferto remove excess vIGF2 peptide. Peak fractions of vIGF2-rhGAA werepooled and concentrated using Amicon Ultra with a 30 kDa nominalmolecular weight cut-off cellulose acetate membrane. vIGF2-rhGAA wascharacterized by receptor plate binding assays to determine whether thehydrazone-linked vIGF2 peptide would increase rhGAA binding to theintended IGF2/CI-MPR receptor. As shown in FIG. 4B, vIGF2-rhGAA boundthe IGF2/CI-MPR receptor significantly better than unconjugated rhGAAand correlated with substantially better cellular uptake of theexogenous lysosomal enzyme in a skeletal muscle cell model (FIG. 5A).These results show the functional importance of improved receptorbinding for enhanced cellular uptake of the exogenous therapeutic drugin target muscle cells. Western blot analysis of muscle cell lysatesshow that internalized vIGF2-rhGAA was delivered to lysosomes wherevIGF2 peptide was removed and rhGAA was processed normally as observedfor the unconjugated rhGAA enzyme (FIG. 5B). These results were expectedsince IGF2 peptide was previously reported to be naturally degraded byresident lysosomal proteases. Importantly, the western blot dataindicate that chemically coupled vIGF2 peptide was removed and did notimpede GAA processing in lysosomes. (GAA processing is required for highaffinity binding and optimal GAA enzyme kinetics for hydrolyzing thenatural glycogen substrate).

These collective data therefore confirm that this alternative chemicalconjugation can be utilized for generating vIGF2-rhGAA enzyme conjugateswhich have substantially better binding to the intended IGF2/CI-MPRreceptor for improved cellular uptake and delivery of therapeutic drugto lysosomes of target cells. This alternative chemicalmodification/conjugation procedure generates stable intermediatecomponents that can be stored indefinitely prior to final chemicalconjugation and purification. Different batches of stable intermediatescan also be pooled to form larger batches for the final conjugationreaction. The methods described herein therefore represent importantadvancements to enable scale up chemical conjugation process forgenerating improved ERTs.

Example 5

In addition to utilizing tBoc-protected hydrazide crosslinkers formodification of vIGF2 peptide, other crosslinkers with differentprotection groups can also be utilized. We have chemically synthesized abifunctional crosslinker containing a methylbenzyloxy carbonyl(BOM)-protected hydrazide (NHS-PEG4-BOM-hydrazide) for introducing anovel hydrazide group to the N-terminus of vIGF2 peptide. After removalof the BOM protection group, the chemically reactive hydrazide group isexposed for subsequent conjugation to benzaldehyde-modified lysosomalenzymes via a resultant hydrazone linkage as follows. Lyophilized,purified vIGF2 peptide was reconstituted at 0.6 mg/ml in 50 mM sodiumphosphate (pH 7.5)/50 mM NaCl/0.05% polysorbate-80 and incubated with a20-fold molar excess of NHS-PEG4-BOM-hydrazide at ambient temperature tointroduce a novel BOM-protected hydrazide group at the N-terminus. Thereaction was monitored by C4 RP-HPLC to assess the progression andextent of modification of vIGF2. A significant shift (increase) in theretention time is observed upon chemical modification of vIGF2 peptideto attach PEG4-BOM-hydrazide (FIG. 6, middle panel). After completion ofchemical modification of vIGF2 peptide (typically by 2 hrs),BOM-hydrazide-modified vIGF2 peptide was purified by preparative C4RP-HPLC to remove excess crosslinker and reaction byproducts andlyophilized to remove volatile solvents. The dried peptide was thenreconstituted in 2% TFA (in dH₂O) and incubated at ambient temperaturefor up to 72 hrs to remove the BOM protection group and expose hydrazidegroup for subsequent conjugation to lysosomal enzymes. The progressionof BOM de-protection was also monitored by C4 RP-HPLC and typicallyshown to decrease the retention time of peptide upon removal of BOM tonear that of the starting, unmodified vIGF2 peptide (FIG. 6, bottompanel). Complete de-protection of BOM group typically requiredapproximately 60 hrs under these experimental conditions. BOMde-protected hydrazide-modified vIGF2 was then purified by preparativeC4 RP-HPLC, lyophilized and stored dry until chemical conjugation tobenzaldehyde-modified rhGAA.

De-protected hydrazide-modified vIGF2 peptide can then be chemicallyconjugated to benzaldehyde-modified rhGAA as described above in Example2 for tBoc-deprotected hydrazide-vIGF2 peptide. These data show thatvIGF2 peptide can be chemically modified with bifunctional crosslinkerscontaining various protected hydrazide groups and after removal ofprotecting group, the same chemically reactive hydrazide was generatedfor conjugating vIGF2 peptide to benzaldehyde-modified lysosomalenzymes.

Example 6

Benzaldehyde-modified rhGAA can also be utilized for chemicalconjugation to aminooxy-modified vIGF2 peptide via a very stable oximelinkage as follows. Lyophilized, purified vIGF2 peptide wasreconstituted at 0.6 mg/ml in 50 mM sodium phosphate (pH 7.5)/50 mMNaCl/0.05% polysorbate-80 and incubated with a 20-fold molar excess ofPhthalimidooxy-PEG12-NHS ester (Quanta Biodesign) at ambient temperatureto introduce a novel phthalimidooxy group at the N-terminus. Aftercompletion of chemical modification of vIGF2 peptide (typically by 2hrs), phthalimidooxy-modified vIGF2 peptide was then incubated with 0.5Mhydrazine at ambient temperature for 30-60 min to convert theintermediate phthalimidooxy group to the desired aminooxy group. C4RP-HPLC was utilized for monitoring the chemical modification reactionand the conversion of the phthalimidooxy group to aminooxy group. Asshown in FIG. 7A, aminooxy-modified vIGF2 peptide had a slightly longerretention time on C4 RP-HPLC as compared to unmodified vIGF2 peptide.Aminooxy-modified vIGF2 peptide is acidified with TFA and then purifiedby preparative C4 RP-HPLC to remove excess crosslinker and reactionbyproducts and lyophilized to remove volatile solvents. The driedaminooxy-modified hydrazide vIGF2 peptide was used directly forchemically conjugation to benzaldehyde-modified rhGAA via a resultantoxime linkage. Briefly, the protein concentration ofbenzaldehyde-modified rhGAA was typically adjusted to 4-5 mg/ml with 50mM sodium phosphate (pH 6.0)/100 mM NaCl/0.05% polysorbate-80 buffer andthe enzyme was added directly to the dry aminooxy-modified vIGF2 peptide(at a molar ratio of 1 mole rhGAA:4 moles vIGF2 peptide) and incubatedat about 20° C. for approximately 16 hours. vIGF2 peptide-conjugatedrhGAA was then purified by size exclusion chromatography in 50 mM sodiumphosphate (pH 6.0)/100 mM NaCl/2% mannitol/0.05% polysorbate-80 bufferto remove excess vIGF2 peptide as described before.

vIGF2-rhGAA was characterized by receptor plate binding assays todetermine whether the oxime-linked vIGF2 peptide would increase rhGAAbinding to the intended IGF2/CI-MPR receptor. As shown in FIG. 7B,vIGF2-rhGAA bound the IGF2/CI-MPR receptor substantially better thanunconjugated rhGAA. In addition, the oxime linkage is very stable andensures that vIGF2 peptide remains conjugated to rhGAA and is onlyremoved upon its degradation in lysosomes after delivery of therapeuticdrug.

Example 7

vIGF2 peptide can also be chemically conjugated to lysosomal enzymes viaan oxime linkage using tBoc-protected aminooxy bifunctional crosslinker.This approach yields aminooxy-modified vIGF2 peptide for chemicalconjugation to benzaldehyde-modified lysosomal enzymes without requiringchemical conversion using potentially dangerous hydrazine. Briefly,lyophilized, purified vIGF2 peptide was reconstituted at 0.6 mg/ml in 50mM sodium phosphate (pH 7.5)/50 mM NaCl/0.05% polysorbate-80 andincubated with a 20-fold molar excess of the bifunctional crosslinkertBoc-protected aminooxy crosslinker (with either NHS- orpentafluorobenzene group) at ambient temperature to introduce a noveltBoc-protected aminooxy group at the N-terminus and monitored by C4RP-HPLC. As shown in FIG. 8 (middle panel), a significant shift(increase) in the retention time is observed upon chemical modificationof vIGF2 peptide to attach PEGylated tBoc-aminooxy crosslinker (middlepanel). After completion of chemical modification of vIGF2 peptide(typically by 2 hrs), tBoc-aminooxy-modified vIGF2 peptide was purifiedby preparative C4 RP-HPLC to remove excess crosslinker and reactionbyproducts and lyophilized to remove volatile solvents. The driedpeptide was then reconstituted in 2% TFA (in dH₂O) and incubated atambient temperature for up to 72 hrs to remove the tBoc protection groupand expose aminooxy group for subsequent conjugation to lysosomalenzymes. The progression of tBoc de-protection was also monitored by C4RP-HPLC and typically shown to decrease the retention time of peptideupon removal of tBoc to near that of the starting, unmodified vIGF2peptide (FIG. 8, bottom panel). Complete de-protection of tBoc grouptypically required approximately 50 hrs under these experimentalconditions. tBoc de-protected aminooxy-modified vIGF2 was then purifiedby preparative C4 RP-HPLC, lyophilized and stored dry until chemicalconjugation to benzaldehyde-modified lysosomal enzymes.

Example 8

An alternative method to generate oxime linked vIGF2-rhGAA using asingle crosslinker can be achieved by coupling aminooxy-modified vIGF2peptide to chemically oxidized rhGAA as follows. rhGAA was bufferexchanged into 0.1M NaOAc (pH 5.2) via dialysis or size exclusionchromatography (SEC) and the protein concentration was adjusted to 5mg/ml with same buffer. rhGAA was then incubated with 2 mM sodiummeta-periodate at 4° C. for 30 min to oxidize carbohydrates (mostlyterminal sialic acids and galactose) on N-glycans of rhGAA to generatechemically reactive aldehyde groups for chemical conjugation toaminooxy-modified vIGF2 peptide. Chemically oxidized rhGAA was thendialyzed against 0.1M NaOAc (pH 5.2) or subjected to SEC to removeexcess sodium meta-periodate. Oxidized rhGAA (stored at approximately 4mg/ml) was added directly to the dry aminooxy-modified vIGF2 peptide ata molar ratio of 1 mole rhGAA:4 moles vIGF2 peptide and incubated atabout 20° C. for approximately 16 hours to form the resultant oximelinkage. vIGF2 peptide-conjugated rhGAA was then purified by SEC in 50mM sodium phosphate (pH 6.0)/100 mM NaCl/2% mannitol/0.05%polysorbate-80 buffer to remove excess vIGF2 peptide as describedbefore.

vIGF2-rhGAA was characterized by receptor plate binding assays todetermine whether the oxime-linked vIGF2 peptide would increase rhGAAbinding to the intended IGF2/CI-MPR receptor. As shown in FIG. 9,vIGF2-rhGAA bound the IGF2/CI-MPR receptor substantially better thanunconjugated rhGAA. The improved receptor binding was dependent on thepresence of vIGF2 since oxidized rhGAA (lacking vIGF2 peptide) was shownto have similar binding as the starting unconjugated rhGAA enzyme. Thereceptor binding data also suggest that more vIGF2 peptides wereconjugated to chemically oxidized rhGAA than SFB (benzaldehyde)-modifiedrhGAA. Since there are more terminal carbohydrates on N-glycans whichare fully accessible and can be chemically oxidized to form aldehydes,there is high potential for conjugation to more vIGF2 peptides ascompared to SFB-modified rhGAA (which introduces an average of 2benzaldehyde groups for coupling to vIGF2 peptide using the describedprocedure herein). These results therefore confirm thataminooxy-modified vIGF2 peptide can be utilized for coupling toaldehyde-containing lysosomal enzymes for improved binding to theintended IGF2/CI-MPR receptor for better delivery of therapeutic drug.

1. A method of chemical conjugation comprising: a. contacting alysosomal enzyme with a first crosslinking agent to introduce aldehydegroups; b. contacting a lysosomal targeting peptide with a secondcrosslinking agent to introduce a hydrazide group at the N-terminalresidue; c. contacting the lysosomal enzyme with aldehyde groups of awith the lysosomal targeting peptide with a hydrazide group at theN-terminal residue of b; and d. forming a lysosomal enzyme-lysosomaltargeting peptide conjugate.
 2. The method of claim 1 wherein thelysosomal targeting peptide comprises variant insulin-like growth factor2 (vIGF2).
 3. The method of claim 1, wherein the first crosslinkingagent comprises N-succinimidyl-4-formylbenzamide (S-4FB).
 4. The methodof claim 1, wherein the second crosslinking agent comprisesN-tert-butoxycarbonyl (tBoc)-protected hydrazide crosslinker(NHS-PEG4-tBoc-hydrazide).
 5. The method of claim 1, wherein the secondcrosslinking agent is a bifunctional crosslinker comprising acetone-,dimethylbenzyloxycarbonyl-, trimethylbenzyloxycarbonyl-protectedhydrazide groups, or any combination thereof.
 6. The method of claim 5,wherein the bifunctional crosslinker comprises Acetone-protectedNHS-PEG₄-hydrazide, Dimethylbenzylcarbonyl-protected NHS-PEG₄-hydrazide,Trimethylbenzyloxycarbonyl-protected NHS-PEG₄-hydrazide, or anycombination thereof.
 7. The method of claim 1, wherein the lysosomalenzyme comprises acid α-glucosidase (rhGAA), acid α-galactosidase A(GLA), acid β-glucuronidase (GUS), acid α-iduronidase A (IduA), acididuronidate 2-sulfatase (I2S), β-hexosaminidase A (HexA),β-hexosaminidase B (HexB), acid α-mannosidase A, β-glucocerebrosidase(GlcCerase), acid lipase (LPA), or combination thereof.
 8. The method ofclaim 1, wherein the lysosomal enzyme is human.
 9. The method of claim1, wherein the lysosomal targeting peptide with a hydrazide group at theN-terminal residue is added in c at about four-fold molar excess. 10.The method of claim 1, wherein aniline is added in c.
 11. A bifunctionalcrosslinker comprising acetone-, dimethylbenzyloxycarbonyl-,trimethylbenzyloxycarbonyl-protected hydrazide groups, or anycombination thereof.
 12. The bifunctional crosslinker of claim 11wherein the bifunctional crosslinker comprises Acetone-protectedNHS-PFG₄-hydrazide, Dimethylbenzylcarbonyl-protected NHS-PEG₄-hydrazide,Trimethylbenzyloxycarbonyl-protected NHS-PEG₄-hydrazide, or anycombination thereof.
 13. A modified lysosomal targeting peptidecomprising a protected hydrazide group.
 14. The modified lysosomaltargeting peptide of claim 12 wherein the modified lysosomal targetingpeptide comprises modified vIGF2 peptide.
 15. The modified lysosomaltargeting peptide of claim 13, wherein the hydrazide group is protectedby acetone-, dimethylbenzyloxycarbonyl-,trimethylbenzyloxycarbonyl-groups, or any combination thereof.
 16. Amethod of making a modified lysosomal targeting peptide, comprising:contacting a lysosomal targeting peptide with a crosslinking agent tointroduce a hydrazide group at the N-terminal residue.
 17. The method ofclaim 16 wherein the lysosomal targeting peptide is vIGF2.
 18. Themethod of claim 16, wherein the crosslinking agent comprisesN-tert-butoxycarhonyl (tBoc)-protected hydrazide crosslinker(NHS-PEG4-tBoc-hydrazide).
 19. The method of claim 16, wherein thecrosslinking agent comprises acetone-, dimethylbenzyloxycarbonyl-,trimethylbenzyloxycarbonyl-protected hydrazide groups, or anycombination thereof.
 20. The method of claim 16, wherein thebifunctional crosslinker comprises Acetone-protected NHS-PEG₄-hydrazide,Dimethylbenzylcarbonyl-protected NHS-PEG₄-hydrazide,Trimethylbenzyloxycarbonyl-protected NHS-PEG₄-hydrazide, or anycombination thereof.
 21. The method of claim 16, further comprisingdeprotecting the protected hydrazide groups of the modified lysosomaltargeting peptide.
 22. A method of linking one or more lysosomaltargeting peptides to a lysosomal enzyme comprising: a. deprotecting ahydrazide-modified lysosomal targeting peptide in solution to form adeprotected hydrazide-modified lysosomal targeting peptide; b. andlinking at least one deprotected hydrazine modified lysosomal targeting,peptide to a lysosomal enzyme.
 23. The method of claim 22 wherein thelysosomal targeting peptide is vIGF2.
 24. The method according to claim22, wherein the lysosomal enzyme comprises acid α-glucosidase (rhGAA),acid α-galactosidase A (GLA), acid β-glucuronidase (GUS), acidα-iduronidase A (IduA), acid iduronidate 2-sulfatase (I2Sβ-hexosaminidase A (HexA), β-hexosaminidase B (HexB), acid α-mannosidaseA, β-glucocerebrosidase (GlcCerase), acid lipase (LPA), or ancombination thereof.
 25. A lysosomal enzyme-lysosomal targeting peptideconjugate, comprising: a lysosomal enzyme crosslinked to one or morelysosomal targeting peptides via one or more bifunctional crosslinkers.26. The method of claim 25 wherein the bifunctional crosslinkerscomprise Acetone-protected NHS-PEG₄-hydrazide,Dimethylbenzylcarbonyl-protected NHS-PEG₄-hydrazide,Triemethylbenzyloxycarbonyl-protected NHS-PEG₄-hydrazide,N-succinimidyl-4-formylbenzamide (S-4FB), or any combination thereof.27. The method of claim 25, wherein the lysosomal enzyme comprises acidα-glucosidase (rhGAA), acid α-galactosidase A (GLA), acidβ-glucuronidase (GUS), acid α-iduronidase A (IduA), acid iduronidate2-sulfatase (I2S), β-hexosaminidase A (HexA), β-hexosaminidase B (HexB), acid α-mannosidase A, β-glucocerebrosidase (GlcCerase), acid lipase(LPA), or any combination thereof.
 28. The method of claim 25, whereinthe lysosomal targeting peptide comprises vIGF2.